Age-related macular degeneration (AMD) is the leading cause of untreatable vision loss in elderly Americans (Klein et al., Invest. Ophthalmol. Vis. Sci. 36:182-191 (1995)). The initial stages of the disease are neither well understood nor currently treatable. The photoreceptors of the retina comprise the rods and cones, each of which is a specialized sensory cell, a bipolar neuron. Each is composed of an inner and an outer region. The cone's outer segment, like that of adjacent rod photoreceptors, consists of a series of stacked cell membranes that are rich in photosensitive pigments. The distal tips of the rod outer segments are intimately associated with the outermost layer of the retina, the pigment epithelium (RPE). The rod outer segments are in a continuous state of flux, wherein new stacks of membrane are added at the base of the outer segment, and old, worn-out stacks of membrane are shed from its distal tip. The shed rhodopsin-laden segments are phagocytosed by cells of the retinal pigment epithelium (RPE) and engulfed by lysosomes, becoming residual bodies in the cytoplasm of the epithelial cells. Daily phagocytosis of spent photoreceptor outer segments is a critical maintenance function performed by the RPE to preserve vision. Aging retinal pigment epithelium (RPE) accumulates lipofuscin, which includes N-retinylidene-N-retinylethanolamine (A2E) as the major autofluorescent component. Additional components include partially degraded lipids and proteins of either photoreceptor or RPE origin which can act as precursors and combine into the lipofuscin complex.
A2E is localized to lysosomes in cultured RPE, as well as in human RPE in situ. Thus, one of the earliest characteristics of the disorder is the accumulation of lipofuscin in the RPE (Feeney-Burns et al., Am. J. Ophthalmol. 90:783-791 (1980); Feeney et al., Invest Ophthalmol Vis. Sci. 17:583-600 (1978)). A2E, a primary constituent of lipofuscin (Eldred et al., Nature. 361:724-726, 1993.)), undermines lysosomal organelles in several ways including by elevating lysosomal pH (pHL) (Eldred et al., Gerontol. 2:15-28 (1995); Holz et al., Invest Ophthalmol Vis. Sci. 40:737-743 (1999)). As key lysosomal enzymes act optimally in a narrow range of acidic environments, an increase in pHL reduces their degradative ability. Because of the circadian rhythm of RPE phagocytosis in the eye, a delay in lipid degradation results in a build up of undigested material in RPE after 24 hours. A consequent accumulation of undigested material compromises RPE cells and appears to hasten the development of AMD. In this regard, the restoration of an optimal acidic environment to lysosomes could enhance enzyme activity and slow or stop the progression of AMD.
Dry AMD is characterized by the failure of multiple systems in the posterior eye and is associated with the accumulation of abnormal deposits within and upon Bruch's membrane (Moore et al., Invest Ophthalmol Vis. Sci. 36:1290-1297 (1995)), which separates the blood vessels of the choriod from the RPE layer. The RPE sends metabolic waste from the photoreceptors across Bruch's membrane to the choroid. The Bruch's membrane allows 2-way transit; in for nutrients and out for waste. Thus, Bruch's membrane's vital function is to supply the RPE and outer part of the sensory retina with all of their nutritional needs. However, as Bruch's membrane thickens and gets clogged with age, the transport of metabolites is decreased. This may lead to the formation of drusen, which can be seen in the eye as yellow-gray nodules located between the RPE and Bruch's membrane in age-related macular degeneration (Kliffen et al., Microsc Res Tech. 36:106-122 (1997); Cousins et al., In Macular Degeneration Eds. Penfold & Provis, Springer-Verlag, New York, pp. 167-200, (2005)). Drusen deposits vary in size and may exist in a variety of forms, from soft to calcified. With increased drusen formation the RPE are gradually thinned and begin to lose their functionality. While drusen formation is not necessarily the cause of dry ARMD, it does provide evidence of an unhealthy RPE. There is also a build up of deposits (Basal Linear Deposits or BLinD and Basal Laminar Deposits BLamD) on and within the membrane. Consequently, the retina, which depends on the RPE for its vitality, may be affected and vision problems arise.
While the initial triggers for these changes are not certain, decline in the hydraulic conductivity of Bruch's membrane, decreased choroidal perfusion (Lutty et al., Mol. Vis. 5:35 (1999)), environmental and immunologic injury (Beatty et al., Surv. Ophthalmol. 45:115-134 (2000); Zhang et al., J. Cell. Sci. 116:1915-1923 (2003)), genetic defects (Kuehn et al., J. Am. Med. Ass. 293:1841-1845 (2005); Ambati et al., Nature. Med. 9:1390-1397 (2003)), and other degenerative diseases (Johnson et al., Proc. Nat. Acad. Sci. USA 99:11830-11835 (2002); Mullins et al., FASEB. J. 14:835-846 (2000)) may all contribute to the development of the pathology. The identification of lysozyme C and oxidation products of docosahexaenoate in material present between Bruch's membrane and the RPE suggests that the extrusion of material from the lipofuscin-laden RPE contributes to sub-retinal deposit formation (Young et al., Surv. Ophthalmol. 31:291-306 (1987); Crabb et al., Proc. Nat. Acad. Sci. USA. 99: 14682-14687 (2002)). The correlation between RPE lipofuscin levels and those retinal regions showing the highest degree of atrophy supports the growing concept that lipofuscin is not just an indicator of disease, but rather, is itself a causal factor von Ruckmann et al., Graefes Arch. Clin. Exp. Ophthalmol. 237:1-9 (1999); Roth et al., Graefes. Arch. Clin. Exp. Ophthalmol. 242:710-716 (2004), suggesting that a reduction in the rate of lipofuscin formation and an enhancement in lysosomal degradative capacity will slow or stop the progression of AMD before substantial degeneration has occurred.
Lipofuscin in the RPE is primarily derived from incomplete digestion of phagocytosed photoreceptor outer segments (Young et al., Surv. Ophthalmol. 31:291-306 (1987); Eldred., In The Retinal Pigment Epithelium, Eds. Marmor & Wolfensberger, Oxford, University Press, New York, pp. 651-668, (1998)), with rates of formation reduced when photoreceptor activity is diminished (Katz et al., Exp. Eye. Res. 43:561-573 (1986); Sparrow et al., Exp. Eye. Res. 80:595-606 (2005)). A2E is a key component of RPE lipofuscin, with A2PE, iso-A2E and other related forms present (Eldred et al., supra, 1993; (Mata et al., Proc. Nat. Acad. Sci. USA 97:7154-7159 (2000)).
A2E has been identified in post-mortem eyes from elderly subjects, while levels are substantially elevated in Stargardt's disease, characterized by early-onset macular degeneration (Mata et al., supra, 2000). The disease is associated with mutations in the ABCA4 (ABCR) gene, whose product transports a phospholipid conjugate of all-trans-retinaldehyde out of the intradisk space of the photoreceptors (Allikmets et al., Nature. Gen. 15:236-246 (1997); Sun et al., Nature. Gen. 17:15-16 (1997)). The accumulation of substrate resulting from the transport failure leads to formation of A2PE, which is subsequently delivered to the RPE after the phagocytosis of the outer segments (Sun et al., J. Biol. Chem. 274:8269-8281 (1999)). A2PE is cleaved to A2E in the RPE, with small amounts of spontaneous isomerization to iso-A2E occurring (Parish et al., Proc. Nat. Acad. Sci. USA 95:14609-1413 (1998); Ben-Shabat et al., J. Biol. Chem. 277:7183-7190 (2002)). Measurements from ABCA4−/− mice, developed by Travis and colleagues, have demonstrated that A2E levels are greatly enhanced in the RPE of ABCA4 mutant mice, consistent with the elevated levels of A2E in patients with Stargardt's disease (Mata et al., supra, 2000). In a rate-determining step in the visual cycle, retinaldehyde is reduced to retinol by the enzyme retinol dehydrogenase located in the photoreceptor outer segment. Thus, only the retinaldehyde that escapes conversion to retinol can react with phosphatidylethanolamine, and enter the A2E biosynthetic pathway to generate A2E in a multistep process.
The above-noted localization of A2E predominantly to lysosomes and late endosomes of RPE cells in vitro and in situ, is consistent with the phagolysosomal origins of lipofuscin granules (Holz et al., supra, 1999; Finnemann et al., Proc. Natl. Acad. Sci. USA 99:3842-3847 (2002)). As lysosomal organelles in the RPE degrade phagocytosed outer segments, the accumulation of undigested material of outer segment origin in AMD is consistent with a lysosomal dysfunction. Addition of A2E to cultured cells reduces the lysosomal degradation of photoreceptor outer segment lipids (Finnemann et al., supra, 2002), and decreases the pH-dependent protein degradation attributed to lysosomal enzymes (Holz et al., supra, 1999).
The mechanisms by which A2E causes lysosomal damage are influenced by levels of light and A2E itself. At high concentrations, the amphiphilic structure leads to a detergent-like insertion of A2E into the lipid bilayer, with consequent loss of membrane integrity and leakage of lysosomal enzymes (Eldred et al., supra, 1993; Sparrow et al., Invest. Ophthalmol. Vis. Sci. 40:2988-2995 (1999); Schutt et al., Graefes. Arch. Clin. Exp. Ophthalmol. 240:983-988 (2002)). Low-wavelength light can oxidize lipofuscin and A2E into toxic forms, which rapidly lead to cell death (Sparrow et al., supra, 2005; Sparrow et al., Invest. Ophthalmol. Vis. Sci. 41:1981-1989 (2000)). The direct effect on degradative lysosomal enzymes is also dependent on light. While lipofuscin directly decreases the activity of several lysosomal enzymes removed from lysosomes when exposed to light, it had little effect on their activity in the dark (Shamsi et al., Invest. Ophthalmol. Vis. Sci. 42:3041-3046 (2001)). The lack of direct effects on lysosomal enzymes in the absence of light treatment has been confirmed by Bermann et al., Exp Eye Res. 72:191-195 (2001).
Conversely, however, indirect effects are likely, since A2E interferes with the function of the lysosomal vH+ATPase proton pump (Bergmann et al., FASEB. J. 18:562-564 (2004)), and low levels of A2E increased lysosomal pH (Holz et al., supra, 1999). The detected lysosomal pH change indicated that A2E could reduce enzyme effectiveness by alkalinizing the lysosomes. Yet, because this pH-dependent effect occurred at low levels of A2E that had little effect on membrane leakage, the alkalinization apparently preceded acute disruption of membrane integrity.
The modification and degradation of material by lysosomes is essential for cellular function. Lysosomes are characterized by their low pH (4.5-5.0), with optimal enzyme activity dependent on vesicle pH (Geisow et al., Exp. Cell. Res. 150:36-46 (1984)). Lysosomes are thought to acidify when positively charged hydrogen ions are pumped across the membrane by an H+-ATPase pump, but the build up of charge limits the degree of acidification. The charge imbalance is overcome by the movement of negatively charged chloride ions into the lysosome through a Cl− channel. Thus, agents that cause the Cl− channel to open, lead to further acidification of the lysosome.
The degradation of outer segment proteins of the photoreceptor is primarily mediated by the aspartic protease cathepsin D (Hayasaka et al., J. Biochem. 78:1365-1367 (1975)). While its pKA varies with substrate, the degradative activity of cathepsin D is generally optimum near pH 4, and falls below 20% of maximum at pH>5.0 (Barrett, In Protinases in Mammalian Cells and Tissues, Elsiver/North-Hollard, Biomedical. Press, New York, pp. 220-224 (1977)). Rats treated with chloroquine, which is known to alkalinize lysosomes (Krogstad et al., Am. J. Trop. Med. Hyg. 36:213-220 (1987)), doubled the number of outer segment-derived lysosome-associated organelles in the RPE (Mahon et al., Curr. Eye. Res. 28:277-284 (2004)), leading to the finding that lysosomal alkalization by A2E contributes to the accumulation of lipofuscin in the AMD. However, pharmacologic restoration in a disorder that progresses over decades can be fully realized only when the mechanisms controlling lysosomal pH are understood. Thus, the present invention serves an important function by meeting this need.
Lysosomal vesicle acidification is regulated by a series of membrane proteins, with proton delivery to lysosomes and late endosomes primarily mediated by the vacuolar proton pump (vH+ATPase). The transport of protons by vH+ATPases creates both a proton gradient and an electrical potential across vesicular membranes (Schneider D L., J. Biol. Chem. 256: 3858-3864, 1981; (Faundez et al., Science's Stke. 233:re8 (2004)). While the activity of vH++ATPase in these vesicles is frequently constitutive, the conductance through CV channels is regulated (Hara-Chikuma et al., J. Biol. Chem. 280:1241-1247 (2005); Barasch et al., J. Cell. Biol. 107: 2137-2147 (1988); Sonawane et al., J. Biol. Chem. 277:5506-5513 (2002)).
Thus, a need has remained in the art, until the present invention, to find a way to slow the progression of AMD, particularly by regulating the acidity of the lysosomes within the RPE cells.